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. Author manuscript; available in PMC: 2012 Oct 1.
Published in final edited form as: J Vasc Surg. 2011 Jul 31;54(4):1117–1123.e1. doi: 10.1016/j.jvs.2011.04.037

Inhibition of Transforming Growth Factor-β Restores Endothelial Thromboresistance in Vein Grafts

Navin K Kapur a,c, Ce Bian a, Edward Lin a, Clayton B Deming a, Jason L Sperry b, Baranda S Hansen a, Nikolaos Kakouros a, Jeffrey J Rade a
PMCID: PMC3188354  NIHMSID: NIHMS291198  PMID: 21803524

Abstract

Background

Thrombosis is a major cause of the early failure of vein grafts (VGs) implanted during peripheral and coronary arterial bypass surgeries. Endothelial expression of thrombomodulin (TM), a key constituent of the protein C anticoagulant pathway, is markedly suppressed in VG after implantation and contributes to local thrombus formation. While stretch-induced paracrine release of transforming growth factor-β (TGF-β) is known to negatively regulate TM expression in heart tissue, its role in regulating TM expression in VGs remains unknown.

Methods

Changes in relative mRNA expression of major TGF-β isoforms were measured by qPCR in cultured human saphenous vein smooth muscle cells (HSVSMC) subjected to cyclic stretch. To determine the effects of paracrine release of TGF-β on endothelial TM mRNA expression, human saphenous vein endothelial cells (HSVEC) were co-cultured with stretched HSVSMC in the presence of 1D11, a pan-neutralizing TGF-β antibody, or 13C4, an isotype-control antibody. Groups of rabbits were then administered 1D11 or 13C4 and underwent interpositional grafting of jugular vein segments into the carotid circulation. The effect of TGF-β inhibition on TM gene expression was measured by qPCR, protein C activating capacity and local thrombus formation were measured by in situ chromogenic substrate assays, and VG remodeling was assessed by digital morphometry.

Results

Cyclic stretch induced TGF-β1 expression in HSVSMC by 1.9±0.2-fold, P<.001 without significant chang in the expressions of TGF-β2 and TGF-β3. Paracrine release of TGF-β1 by stretched HSVSMC inhibited TM expression in stationary HSVEC placed in co-culture by 57±12%, P=.03, an effect that was abolished in the presence of 1D11. Similarly, TGF-β1 was the predominant isoform induced in rabbit VGs 7 days after implantation (3.5±0.4-fold induction, P<.001). TGF-β1 protein expression localized predominantly to the developing neointima and coincided with marked suppression of endothelial TM expression (16±2% of vein controls, P<.03), a reduction in situ APC-generating capacity (53±9% of vein controls, P=.001) and increased local thrombus formation (3.7±0.8-fold increase over vein controls, P<.01). External stenting of VGs to limit vessel distension significantly reduced TGF-β1 induction and TM downregulation. Systemic administration of 1D11 also effectively prevented TM downregulation, preserved activated protein C-generating capacity and reduced local thrombus in rabbit VGs without observable effect on neointima formation and other morphometric parameters 6 weeks after implantation.

Conclusion

TM downregulation in VGs is mediated by paracrine release of TGF-β1 caused by pressure-induced vessel stretch. Systemic administration of an anti-TGF-β antibody effectively prevented TM downregulation and preserved local thromboresistance without negative effect on VG remodeling.

INTRODUCTION

Autologous saphenous vein grafts (VGs) remain widely used conduits for both peripheral and coronary arterial bypass graft surgeries. Compared to arterial conduits, VGs are particularly susceptible to thrombotic occlusion which limit the clinical efficacy of these procedures.1 Despite the routine use of antiplatelet agents, occlusion rates as high as 20–30% within the first post-operative year continue to be observed for VGs implanted into both the peripheral and coronary arterial circulations.2–4

Thrombomodulin (TM) is a transmembrane endothelial cell protein essential to maintaining vascular thromboresistance.5 It binds and alters the active site specificity of thrombin, preventing both thrombin-mediate cleavage of fibrinogen and activation of cellular thrombin receptors while enabling thrombin-mediated enzymatic activation of circulating protein C. Activated protein C (APC) degrades Factors Va and VIIIa of the coagulation cascade, thereby inhibiting further thrombin formation.

We previously found that TM expression was profoundly reduced in rabbit VGs in the weeks following implantation, resulting in a reduced ability of the graft endothelium to generate APC that directly resulted in increased local thrombus formation.6 Subsequent studies identified pressure-induced stretch as the critical hemodynamic stimulus negatively regulating TM gene expression in VG.7 The ability of stretch to negatively regulate endothelial TM expression and function is not confined only to VGs but appears to be a more general biologic response. In a rat model of acute heart failure, we similarly found that TM expression by atrial endocardial endothelial cells was downregulated by pressure overload-induced stretch.8 In the heart, stretch-induced paracrine release of transforming growth factor-β (TGF-β) by cardiac connective tissue was identified as the primary mediator of TM gene downregulation and impaired APC generating-capacity in the overlying endocardial endothelial cells.

The aim of the present study was to investigate the relationship between TGF-β induction and TM regulation in VGs. We first characterized TGF-β isoform induction by vascular stretch in vitro and in vivo. We then explored the utility of systemic administration of a neutralizing anti-TGF-β antibody to restore TM expression, APC generating capacity and reduce local thrombus formation. Finally, we investigated the effect of TGF-β inhibition on the development of VG remodeling and neointimal hyperplasia.

MATERIALS AND METHODS

Cell culture

Surplus saphenous vein segmentswere obtained from patients at the time cardiac bypass surgery with approval of the Johns Hopkins School of Medicine and Tufts Medical Center offices of Human Subjects Research. Human saphenous endothelial cells were isolated by brushing the luminal surface of divided vein segments with a sterile cotton swab that was then used to inoculate tissue culture plates containing EGM-2 culture medium (Cambrex, Walkersville, MD). Once established in culture, endothelial cells were subjected to fluorescence activated cell sorting to greater than 99.9% purity with an anti-CD31 monoclonal antibody (#555445; BD Biosciences, San Diego, CA) as previously described.8 Human saphenous vein smooth muscle cells (HSVSMC) were isolated using the outgrowth technique9, cultured in low-glucose DMEM (Invitrogen, Carlsbad, CA) supplemented with 1% L-glutamine, 10% bovine growth serum (HyClone, Logan, UT) and antibiotics then characterized by positive staining for smooth muscle cell actin.

In vitro stretch experiments

Cells were cultured in their respective media to near-confluence on type I collagen-coated 6-well Bioflex plates (Flexcell International, Hillsborough, NC) then subjected to cyclic stretch at 1 Hz at 37°C under 5% CO2 for the indicated time with an FX-4000T Tension Plus System (Flexcell International). To determine the effects of paracrine release of TGF-β on endothelial TM expression in human vascular endothelial cells, a previously described co-culture system was employed8 whereby HSVEC were first cultured on 24-mm diameter Transwell inserts with a 0.4 μm pore size (Corning, Corning, NY) in EGM-2 medium. When confluent, the inserts were suspended in the individual wells of Bioflex plates containing confluent HSVSMC in EBM medium (Cambrex) containing 0.5 μg/mL of either 1D11, a TGF-β pan-neutralizing antibody, or 13C4, an isotype control antibody (both generously provided by the Genzyme Corporation, Cambridge, MA). HSVSMC were then subjected to 10% stretch at 1 Hz for 16 hours and TM mRNA expression in stationary HSVEC determined by quantitative PCR.

Rabbit vein graft model

All animal procedures were approved by the Johns Hopkins University Animal Care and Use Committee. Interpositional grafting of jugular vein segments into the carotid circulation was performed in male New Zealand White rabbits (Robinson Services, Clemmons, NC) as previously described.6,7 Immediately before surgery, 3 mg/kg gentamicin and 250 mg cephazolin were administered intravenously for antibiotic prophylaxis. In a group of rabbits, the surgical protocol was modified to prevent pressure-induced vessel distension by externally stenting VGs with a nondistensible polyethylene 23-gauge needle cover as previously described.7 Stents had a measured lumenal diameter of 2.0±0.0 mm and reduced vessel diameter by over 60% compared with unstented vein grafts. For experiments to determine the effects of TGF-β neutralization, rabbits were intravenously administered 10 mg/kg of either the 1D11 or the 13C4 control antibody in normal saline biweekly for the indicated times.

Quantitative PCR

mRNA was extracted from cultured cells and freshly harvested rabbit VGs using Trizol Reagent (Life Technologies). Aliquots of mRNA were reverse-transcribed into cDNA using random hexamer primers (Superscript First-Strand Synthesis System for RT-PCR; Invitrogen) and real-time quantitative PCR (RT-PCR) was performed in triplicate on a 7900HT Sequence Detector (Applied Biosystems) using TaqMan Universal PCR Mastermix (Applied Biosystems) and primers and labeled probes to the appropriate target gene (see Methods Supplement for a full list of primer/probe sequences).

The mean threshold cycle (CT) values for each sample were compared with those of a standard curve derived from serial dilutions of control jugular vein RNA. Gene expression for endothelial markers were normalized to 18S rRNA and CD31.

Western blot and immunohistochemical analyses

Western blotting was performed using anti-TM antibodies (#236 and #2380; American Diagnostica), anti-CD31 antibody (M0823; Dako, Carpinteria, CA) and an anti-TGF-β1 antibody (MAB240; R&D Systems, Minneapolis, MN) as previously described.6 Bands were detected by autoradiography and quantified densitometrically using UN-SCAN-IT software (Silk Scientific, Orem, UT). Immunohistochemical staining was performed on formalin-fixed and paraffin-embedded sections of rabbit vein grafts using an antibodies against human TGF-β1 latent associated peptide (AF-246-NA; R&D Systems), rabbit TM (#236; American Diagnostica) and human PAI-1 (#395G; American Diagnostica) with an appropriate biotinylated secondary antibody and horseradish peroxidase labeled streptavidin (Vector Laboratories; Burlinngame, CA). Peroxidase activity was revealed by either aminoethylcarbazole (Dako) or diaminobenzidine (Vector).

In situ Protein C and thrombin activity assays

APC-generating capacity and local thrombin generation were measured in situ on freshly harvested rabbit vein grafts 7 days after implantation using specific chromogenic substrate assays as previously described.6,8 The rate of substrate conversion was determined spectrophotometrically using a Vmax Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA) and the amounts of APC and thrombin generated per luminal surface area were calculated by comparison to human APC (American Diagnostica) and α-human thrombin (Sigma-Aldrich, St. Louis, MO) standard curves.

Morphometric Analysis

Rabbit vein grafts were harvested, formalin perfusion-fixed and paraffin imbedded 6 weeks after implantation. Three to four Movat-stained transverse sections per graft were digitally imaged with distance calibration using a Spot RT digital camera (Diagnostic Instruments Inc., Sterling Heights, MI) mounted on an Olympus BX60 microscope (Olympus Optical Co., Tokyo, Japan). The lumen, neointima and media areas were masked and measured using SigmaScan Pro 5.0 software (Systat Software, Inc., Richmond, CA) and average neointima thickness was calculated as previously described.6 Mean values for each graft were determined from the separate sections and used to calculate group means.

Statistical Analysis

All data are presented as mean ± SEM. Comparison between two groups is by two-tailed t tests and between multiple groups by one-way ANOVA. Statistically relationships not otherwise indicated are to be assumed non-significant (p >.05).

RESULTS

Paracrine effect of stretch-induced release of TGF-β on endothelial TM expression

As vein segments are subjected pressure-induced stretch when implanted into the arterial circulation, we first characterized the effects of direct mechanical stretch on TM expression in endothelial cells and TGF-β isoform expression in vascular smooth muscle cells in vitro. Human saphenous vein endothelial cells (HSVEC) and smooth muscle cells (HSVSMC) were cultured on collagen-coated silastic membranes and subjected to increasing amounts of cyclic stretch for 16 hours. Direct stretch caused a dose-dependent induction of TM expression in endothelial cells (Figure 1A) as well as of TGF-β1 expression in smooth muscle cells (Figure 1B). We previously showed that stretch of cardiac fibroblasts in vitro and of atrial tissue in vivo causes downregulation of endocardial TM expression via paracrine release of TGF-β.8 To determine if a similar phenomenon might occur in vein grafts, HSVEC were cultured on stationary filters suspended in the media of wells containing HSVSMC subjected to 10% cyclic stretch for 16 hours. In contrast to the direct effects of stretch, TM mRNA expression was significantly reduced in stationary co-cultured HSVEC whilst that of the TGF-β-responsive PAI-1 mRNA expression was markedly increased (Figure 1C). To confirm that endothelial TM downregulation was due to the paracrine effects of TGF-β released by the stretched smooth muscle cells, 1D11, an antibody that neutralizes all three TGF-β isoforms, was added to the culture medium. The 1D11 antibody, but not the isotype control 13C4 antibody that possesses no TGF-β inhibitory properties, effectively prevented TM downregulation and PAI-1 upregulation in the stationary co-cultured endothelial cells (Figure 1D).

Figure 1.

Figure 1

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Effects of cyclic stretch on TM and TGF-β isoform expression. A, HSVEC were subjected to cyclic stretch for 16 hours and the change in TM mRNA expression was determined by quantitative PCR. Values shown are the mean ± SEM of n=4/group. *P <.01 versus unstretched controls. B, HSVSMC were subjected to cyclic stretch for 16 hours and the change in TGF-β isoform mRNA expression was determined by quantitative PCR. Values shown are the mean ± SEM of n=8–11/group. Panel insert is a Western blot showing stretch-induced change in TGF-β1 protein expression. *P <.001 versus unstretched controls. C, HSVEC were co-cultured on stationary filters suspended in the media of wells containing HSVSMC subjected to 10% cyclic stretch for 16 hours and the changes in TM and PAI-1 mRNA expression were determined by quantitative PCR. Values shown are the mean ± SEM of n=3/group. D, HSVEC were co-cultured on stationary filters suspended in the media of wells containing HSVSMC subjected to 10% cyclic stretch for 16 hours in the presence of a pan-neutralizing anti-TGF-β antibody (1D11) or non-neutralizing isotype control antibody (13C4). Changes in TM and PAI-1 mRNA expression were determined by quantitative PCR. Values are the mean ± SEM of n=3/group. *P =.02 versus static controls.

Expression of TGF-β isoforms in vein grafts

TGF-β1 is known to be induced in rabbit VGs within days of implantation and persist for at least 6 months.10 Because little is known about induction of other TGF-β species, the relative mRNA expression of the three TGF-β isoforms was determined in rabbit vein grafts 7 days after implantation into the arterial circulation, at the time of the known nadir in TM mRNA expression.7 Similar to that observed in vitro, TGF-β1 mRNA expression was increased in rabbit vein grafts 3.5-fold (P<.001) compared to ungrafted vein controls while TGF-β2 and TGF-β3 expression was unchanged (Figure 2A). Immunostaining revealed that TGF-β1 and PAI-1 protein expression co-localized predominantly to the developing neointima 7 days after implantation and was associated with loss of TM protein expression in the overlying endothelial cells (Figure 2B, second row panels). In contrast, TGF-β1 and PAI-1 protein expression was minimal in the neointima but localized predominantly in the adventitia 6 weeks after implantation when TM protein expression by the luminal endothelium had returned (Figure 2B, third row panels). To differentiate the effects of vascular stretch from those of pressure on TGF-β1 induction, a group of VGs was externally stented at the time of implantation. Compared to unstented grafts, TGF-β1 mRNA expression was significantly reduced by stenting while TM expression was preserved (Figure 3), indicating that stretch, and not the direct effects of pressure, is the critical hemodynamic stimulus responsible for TGF-β1 upregulation.

Figure 2.

Figure 2

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TGF-β expression in vein grafts. A, Rabbit VGs were harvested 7 days after implantation and relative changes in the mRNA expression of the three major TGF-β isoforms and PAI-1 determined by quantitative PCR. Values are the mean ±SEM of n=4–6/group. *P<.001 and #P=.02 versus ungrafted vein controls. B, Immunostaining for TGF-β1 (left column, brown color), PAI-1 (middle column, brown color) and TM (right column, red color) in rabbit VGs harvested 7 days and 6 weeks after implantation. Staining in a control jugular vein and carotid artery are shown for comparison.

Figure 3.

Figure 3

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Effect of external stenting on TGF-β1 and TM mRNA expression in vein grafts. TGF-β1 and TM mRNA expression was measured in rabbit VGs with and without external stents 3 days after implantation. Values are the mean ± SEM of n=3–4/group. *P<.001 and #P=.02 versus ungrafted vein controls.

Effect of TGF-β inhibition on TM expression and endothelial thromboresistance in vein grafts

To determine if inhibition of TGF-β1 could prevent stretch-induced downregulation of TM and improve VG thromboresistance, rabbits were administered 10 mg/kg intravenously of either the 1D11 or the 13C4 control antibody biweekly beginning prior to VG implantation and continuing for 7 days until graft harvest. Treatment with the TGF-β-neutralizing 1D11 antibody effectively prevented stretch-induced downregulation of TM gene and protein expression (Figures 4A and 4B) and restored APC-generating capacity (Figure 4C) which suppressed local thrombus formation as measured by bound thrombin activity on the graft luminal surface (Figure 4D). Administration of 1D11 also suppressed expression of PAI-1 mRNA which is known to be induced by TGF-β1 (Figure 4E).

Figure 4.

Figure 4

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Effect of TGF-β inhibition on VG thromboresistance. Rabbits were administered 10 mg/kg of the anti-TGF-β 1D11 antibody or the isotype control 13C4 antibody before VG implantation with two subsequent doses prior to graft harvest on day 7. A, TM mRNA expression was determined by quantitative PCR analysis. B, TM protein expression was determined by densitometric analysis of Western blots (a representative Western blot shown in right panel). C, APC generating capacity and D, bound thrombin activity, an indicator of the degree of thrombus formation, were measured in situ using chromogenic assays. E, PAI-1 mRNA expression was determined by quantitative PCR analysis. Values shown are the mean ± SEM for n=6/group. *P <.05 versus 13C4-treated VGs.

Effect of TGF-β inhibition on vein graft neointima formation

The induction of TGF-β1 has been implicated in the remodeling of VGs as they adapt to the arterial circulation.10,11 To determine if antibody-mediated TGF-β inhibition would alter VG remodeling, rabbits were intravenously administered 10 mg/kg of either the anti-TGF-β 1D11 or the13C4 control antibody biweekly beginning prior to VG implantation and continuing for 6 weeks until graft harvest. Despite having beneficial effects on endothelial thromboresistance, administration of the 1D11 antibody had no discernable effects on VG remodeling, including neointima formation (Table 1).

Table 1.

VG morphometry 6 weeks after implantation.

Parameter 1D11 (n=8) 13C4 (n=8) P-value
Lumen Area (mm2) 36.3 ± 11.9 42.8 ±10.4 .65
Neointima Thickness (μm) 101.5 ± 9.1 112.5 ± 10.2 .39
Neointima Area (mm2) 2.1 ±0.4 2.5 ± 0.4 .36
Medial Thickness (μm) 108.4 ± 4.6 114.8 ± 12.4 .62
Medial Area (mm2) 2.3 ± 0.3 2.8 ± 0.6 .39
Lumen radius/Wall thickness Ratio 15.7 ± 2.3 16.9 ± 2.3 .67
Neointima area/EEL area Ratio 0.07 ± 0.01 0.07 ± 0.01 .98
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Abbreviations: EEL, external elastic lamina.

DISCUSSION

The major findings of this study were: 1) TGF-β1 was the predominant TGF-β isoform induced in vitro by cyclic stretch of human vascular fibroblasts and by pressure-induced stretch of rabbit VG in vivo; 2) Paracrine release of TGF-β1 was a potent negative regulator of TM expression and function in rabbit VGs; 3) Systemic administration of a pan-neutralizing anti-TGF-β antibody effectively prevented TM downregulation, restored APC-generating capacity and reduced local thrombus formation in rabbit VGs without altering VG remodeling.

Early VG thrombosis is a multifactorial process resulting from a complex interplay of systemic factors, such as inflammation and hypercoagulation, with factors inherent to the graft, such as impaired blood flow and endothelial dysfunction or injury.1 Of these factors, endothelial dysfunction caused by exposure of vein segments to arterial pressure and flow is the least well understood. We have previously shown that endothelial TM protein expression in VGs is reduced by ~95% within days of implantation and remains depressed for many weeks.6 TM is a key component of the protein C anticoagulant pathway that is critical to maintaining vascular thromboresistance.5 Acquired loss of TM with consequent reduced capacity to activate protein C has been observed in a number of thrombotic conditions such as transplant rejection, atherosclerosis and especially sepsis where administration of recombinant APC is now an approved therapy.12,13 While diminished nitric oxide generation and increased tissue factor expression are among other recognized hemostatic derangements that impair VG thromboresistance in the early post-operative period, the importance of dysfunction of the TM-protein C pathway is evidenced by the ability of targeted restoration of TM expression and APC-generating capacity to reduce local thrombus formation.6,14,15

Modifications to the standard rabbit VG model to alter various hemodynamic parameters performed in our laboratory revealed that TM expression was inversely proportional to wall tension caused by pressure-induced distension of the thin-walled veins.7 Adaption of VGs to the arterial circulation is characterized by medial hypertrophy and neointimal hyperplasia which increase wall thickness and thus reduces wall tension over time. With a decline in wall tension, a concurrent rise in TM expression was observed, though by 6 weeks the level of TM expression remained less than that observed in native arteries. We build upon those findings in the current study by demonstrating that pressure-induced stretch induced the expression and release of TGF-β1 which acts in paracrine fashion to suppress endothelial TM expression. This mechanism is similar to that observed in rat model of acute heart failure where paracrine release of TGF-β inhibited endocardial TM expression in pressure-overloaded left atria.8

The TGF-β family encompasses a wide array of pleiotropic growth factors intimately involved in inflammation, wound repair, angiogenesis and tissue remodeling.16,17 TGF-β1 mRNA expression is known to be induced in VGs in the days after implantation in several different animal models.18–21 In rabbit VGs, TGF-β1 upregulation persists for at least 6 months and is temporally associated with myofibroblast recruitment and matrix deposition that occur during vessel remodeling.10,11,19 Our findings add to these data by demonstrating that TGF-β1 is the dominant TGF-β isoform induced in VGs, that the primary stimulus for induction is vessel stretch rather than the direct effects of pressure, and that TGF-β1 protein localized to the developing neointima early after implantation, concurrent with maximal suppression of endothelial TM expression, but predominated in the adventitia at later time points when endothelial TM expression returned. Although we found that systemic administration of a neutralizing anti-TGF-β antibody effectively prevented the downregulation of TM expression in the VG endothelium, we did not observe any effects on VG remodeling, especially neointima formation. This is in contrast to a prior study where adenovirus-mediated expression of anti-sense RNA directed against TGF-β1 had a modest net effect at reducing neointima formation in a rat VG model, though the adenovirus vector itself in this study appeared to stimulate neointima formation.22

While both TGF-β1 and TGF-β2 are known to be capable of downregulating TM expression in endothelial cells, extremely little is known about the molecular mechanism by which this occurs.23,24 TGF-β1 is initially secreted in an inactive form via association with one of several latency-associated peptides (LAPs) that anchor it to the extracellular matrix in the interstitial space.25 Biologically active TGF-β1 can be released from the latent complex through proteolytic cleavage by matrix metalloproteinases and plasmin or through a conformational change mediated by the binding of thrombospondin or αv integrins to the LAP complex.26 Active TGF-β1 is able to bind specific serine/threonine kinase Type I and Type II receptors causing them to dimerize and phosphorylate a series of downstream effector proteins known as Smads. While most Smad proteins positively regulate TGF-β signal transduction, Smad6 and Smad7 are known to exert inhibitory effects. Only one prior study to date has explored the molecular mechanism by which TGF-β modulates endothelial TM expression. In a study by Sandusky et al., antisense inhibition of Smad7 potentiated the downregulation of TM by TGF-β1 in endothelial cells while antisense inhibition of a splice variant of Smad6, known as Smad6s, unexpectedly blunted TM downregulation.27 Preliminary data in our laboratory indicate that TGF-β1-induced TM downregulation in endothelial cells is predominantly mediated by activation of Smad3 and Smad4, with little observed role of canonical Smad6 (data not shown). It is currently not known what signaling pathways distal to Smad4 activation regulate TM gene transcription, though this remains an area of active investigation.

In summary, our study demonstrates that TM downregulation in VGs is mediated by stretch-induced paracrine release of TGF-β1. Systemic administration of a neutralizing anti-TGF-β antibody effectively restored TM expression and APC-generating capacity which consequently reduced local thrombus formation. Our findings may have implications for preventing VG thrombosis in patients after peripheral and coronary artery bypass surgeries without subjecting them to the bleeding risks of systemic anticoagulation.

Acknowledgments

This study was supported by National Institute Institutes of Health grants HL080142 (to JJR) and HL94909 (to NKK) in addition to material support from the Genzyme Corporation, Cambridge, MA.

The authors wish to thank Kushal Sanghvi, MSc for his technical help.

Footnotes

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